PROJECT SUMMARY Age-related macular degeneration (AMD) is the leading cause of blindness among older persons in developed countries. Yet, there is no effective therapy for the advanced ?dry? form of AMD known as geographic atrophy (GA), in part due to limitations of adequate animal models, many of which have limited lifespan and lack a macula. Nonhuman primates are the only species to possess a macula resembling those in humans and spontaneously develop hallmark features of AMD known as drusen. However, they do not exhibit GA as seen in advanced, atrophic AMD. The goal of this study is to develop a nonhuman primate model of GA using viral expression of optically-inducible reactive oxygen species (ROS)-generating proteins for optogenetic control of mitochondrial oxidative stress in retinal pigment epithelium (RPE). Oxidative stress at the RPE has been implicated as a key factor in the pathogenesis of AMD. However, genetic mouse models such as mice lacking superoxide dismutase (SOD) exhibit widespread RPE cell loss unlike the circumscribed areas of GA in humans, and germline mutations in rodents cannot be readily adapted to nonhuman primates. Genetically-encoded ROS-generating proteins such as KillerRed are phototoxic proteins that can be targeted to specific cell types and produces superoxide upon illumination with specific wavelengths of visible light, allowing temporal and spatial control of ROS. Since the eye provides a natural window for external light, KillerRed expression serves as a powerful tool to enable precise external control of mitochondrial ROS production in the eye. Using an adeno-associated viral (AAV) vector to express inducible ROS-generating proteins in RPE cells, we plan to employ scanning laser ophthalmoscopy (SLO) to trigger localized, titratable ROS production as a unique model of oxidative damage and GA that can be readily quantified using optical coherence tomography (OCT) and fundus autofluorescence (FAF) imaging in vivo, and histological analyses ex vivo. This powerful preclinical model will enable unprecedented preclinical testing of therapeutic agents for advanced atrophic AMD. The specific aims of our study are: Aim 1: To optimize optically-triggered ROS in RPE to create geographic atrophy in C57BL6 mouse eyes We will subretinally inject AAV vectors to express KillerRed in RPE in C57BL6 mice, optimize SLO parameters to trigger focal ROS production, then perform OCT/FAF/OCT-A imaging and histology to measure anatomic changes. Aim 2: To employ optically-triggered ROS in RPE to create geographic atrophy in rhesus macaque eyes We will subretinally deliver AAV vectors to express KillerRed in RPE in macula of rhesus macaques, employ SLO parameters optimized in mice, then employ OCT/FAF imaging and histology to characterize atrophic changes.